The effective diagnosis and treatment of life-threatening fungal infections in humans is a major unmet clinical challenge. Well-annotated genomic sequences of the major human fungal pathogens have been available for a number of years, creating an opportunity to revolutionize medical mycology through the application of systematic genome-wide approaches. However, to be maximally useful, whole-genome sequences need to be coupled with the genome-wide biological resources. The availability of genome-wide knockout and tagged gene collections has been instrumental in dissecting the fundamental biology of the model yeast Saccharomyces cerevisiae. Our premise is that the generation of analogous collections in pathogenic fungi would create powerful experimental tools enabling comprehensive approaches to elucidating the basis of fungal virulence in the mammalian host and to develop drugs. Such tools accelerate research of the community through the application of existing in vivo and in vitro assays to functionally profile the behavior of all genes and proteins in any process of interest. Cryptococcus neoformans is an encapsulated budding yeast that is the most common cause of fungal meningitis. An estimated ~1,000,000 cases result in ~600,000 deaths each year. One-third of deaths in AIDS patients worldwide are the result of Cryptococcal meningoencephalitis. This important fungal pathogen has haploid genetics and homologous recombination, making it an excellent model pathogen and model organism. There are 6967 predicted coding sequences. In the initial period of this grant, we developed an informatics infrastructure, designed and purchased ~70,000 oligonucleotides, recruited/trained personnel and successfully constructed and deposited 4042 gene knockout strains using biolistic (?gene gun?) transformation. We estimate that the current collection covers ~75% of nonessential genes. We have used a portion of this collection to identify new genes required for the production of known virulence factors. Importantly, we have made strains available without restriction to the community via the Fungal Genetic Stock Center. In this amended renewal application, we will complete the Cryptococcus gene deletion collection, perform an additional round of quality- control checks including Southern hybridization, complete our screens for known virulence attributes, obtain reference profiles for fitness during infection, and then leverage our expertise, reagents and infrastructure to construct a strain collection in which each protein-coding gene is dual epitope-tagged, thereby enabling systematic studies of protein function. Completion of this work will have an enormous impact on the field by providing the research community with genomic resources not yet available for any human pathogen.